1. Field of the Invention
The present invention relates to the determination of a peroxidatively active substance in a test sample. More particularly, the invention relates to a composition for such determination which is resistant to possible adverse effects from ascorbic acid which might also be present in the sample.
Many analytical methods are presently available for detecting the presence of peroxidatively active substances in samples such as urine, fecal suspensions, and gastrointestinal contents. Hemoglobin and its derivatives are typical of such "peroxidatively active" substances because they behave in a manner similar to the enzyme peroxidase. Such substances have also been referred to as pseudoperoxidases. Peroxidatively active substances are enzyme-like in that they catalyze the redox reaction between peroxides and such indicator compounds as benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene or similar indicator substances, thereby producing a detectable response such as a color change. Hence, most methods for determining the presence of occult blood in test samples rely on this pseudoperoxidase activity.
2. Description of the Prior Art
Several methods have evolved over the years which rely on enzyme-like catalysis of the peroxidic oxidation of color-forming indicators. Primarily these include wet chemical procedures and "dip-and-read" type reagent-bearing strips. Of the former, a typical example is set forth in Richard M. Henry, et al., Chemical Chemistry Principles and Techniques (Hagerstown, Md.: Harper and Row, 1974), pp. 1124-1125. This procedure involves the use of glacial acetic acid (buffer), diphenylamine (indicator), and hydrogen peroxide. While such wet methods have proven analytical ability, they are nevertheless fraught with obvious shortcomings, not the least of which are poor reagent stability and inadequate sensitivity.
A second method for the determination of peroxidatively active substances, and the one presently preferred by most clinical assayists and analysts, utilizes the so-called "dip-and-read" reagent strips. Typical of such devices are reagent strips manufactured by the Ames Division of Miles Laboratories, Inc. and sold under the name HEMASTIX.RTM.. These comprise, in essence, a porous paper matrix affixed to a plastic strip or handle. The matrix is impregnated with a buffered mixture of an organic hydroperoxide and o-tolidine. Upon immersion in a liquid containing hemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a blue color develops in the matrix, the intensity of which is proportional to the concentration of the peroxidatively active substance in the sample. Thus, by comparing the color developed in the matrix to a standard color chart, the assayist can determine, on a semiquantitative basis, the amount of analyte present in the sample.
The advantages of reagent strips over wet chemistry methods are predominantly twofold; strips are easier to use because neither the preparation of reagents nor the attendant apparatus is required; and greater stability of reagents is afforded, resulting in improved accuracy, sensitivity and economy.
But whether the analysis for the peroxidatively active species be through either alternative, a problem inherent to both exists for which there has to date been no satisfactory solution: interference due to the presence of ascorbate or other reducing agent in the test sample. In the case or urinalysis, for example, the recent popularity of diets which include high dosages of vitamin C (ascorbic acid) has led to serious problems in analyzing for such urine constituents as occult blood, since patients on such diets invariably have atypically elevated levels of urinary ascorbate.
The adverse effects of reducing agents such as ascorbate were recognized as early as 1938. R. Kohn and R. M. Watrous, Journal of Biological Chemistry, 124, 163-168 (1938). That the same problem still plagues this area of analysis is evidenced by a proposal in 1979 that when an occult blood (pseudoperoxidase) analysis in urine is performed, a simultaneous ascorbate analysis be performed in order to gauge the accuracy of the occult blood determination. L. Nielsen, P. J. Jorgensen and A. C. Hansen, Ugeskrift for Laeger, 141, 791-793 (1979).
Although many attempts at removing ascorbate interference with other test systems, such as glucose-sensitive reagents, are reported in the literature, to data no successful attempts have been reported whereby the determination of peroxidatively active substances has been rendered immune to these adverse effects. With the glucose-sensitive systems, approaches range from filtering out ascorbate before it reaches the reagents to utilizing an enzyme to decompose it in situ.
Thus, Canadian Pat. No 844,564, issued on June 16, 1970, to Dahlqvist discloses a device for glucose determination in urine or other media which includes, in addition to a porous portion impregnated with normal glucose-responsive reagents, an additional portion to receive the urine test sample. The sample-receiving portion comprises an ion exchange material, whose singular function in the device is to adsorb any ascorbate which might be present in the urine sample.
Another approach to alleviating ascorbate interference is reflected in U.S. Pat. No. 4,168,205, which issued on Sept. 18, 1979, To Danninger et al. This reference suggests incorporating the enzyme ascorbate oxidase into the test reagent formulation, the theory being that if ascorbate is present in the sample it will be enzymatically oxidized to dehydroascorbate, a compound which does not adversely effect the desired analysis.
U.S. Pat. No. 3,411,887, which issued to Ku on Nov. 19, 1968, describes a way of eliminating ascorbate interference with reagent systems which rely on enzymatic oxidizing substances such as glucose oxidase. An ascorbate "trapping system" is employed. This comprises an "ionizable heavy metal compound which, when in an ionized state possesses an oxidation-reduction potential E.sub.red .degree. between that of the redox indicator dye . . . and that of [ascorbate]". Many metals are cited as examples, including cobalt, iron, mercury and nickel.
In addition to these studies, attention to the ascorbate problem with glucose tests is manifested by:
1. H. Gifford, et al., J. Amer. Med. Assoc., 178, 149-150 (1961).
2. P. O'Gorman, et al., Brit, Med. J., 603-606 (1960).
3. R. Brandt, et al., Clin. Chem, Acta, 51, 103-104 (1974).
4. R. Brandt, et al., Am. J. Clin. Pathol., 68, 592-594 (1977).
Like the above-cited Ku patent other references deal with the complexing and oxidation of ascorbate using cobalt. G. Bragagnolo (Ann. Chim. Applicata, 31, 350-368, 1941) reported that solutions of ascorbic acid were oxidized by air in the presence of cobalt metal. Similar activity has been reported for Co(NH.sub.3).sub.6 Cl.sub.3 by Tomokichi Iwasaki in Journal of the Chemical Society of Japan, 63, 820-826 (1942).
Significantly, although the prior art deals extensively with glucose analysis, it appears bereft of suggestions as to how to solve the ascorbate interference problem with the determination of peroxidatively active substances such as peroxidase and occult blood (hemoglobin). The disclosure in U.S. Pat. No. 3,411,887 (see above) notwithstanding, the prior art unequivocally teaches that metal ions, such as Co(III), are in fact pseudoperoxidases. For example, Co(III) acetate is used commercially to catalytically decompose cumene hydroperoxide. The Merck Index, 9th Ed., Page 311 (1976). A series of Co(III) complexes are reported to catalytically decompose peroxides by Kh. Lohs., Monatsber, Deut. Akad. Wiss, Berlin, 8, 657-659 (1966) (See Chemical Abstracts, 67, 120383z. 1967).
As is stated supra, the present invention deals with improving the present state-of-the-art system for determining peroxidatively active substances. Such systems invariably comprise an organic hydroperoxide and a redox indicator such as o-tolidine or 3,3',5,5'-tetramethyl benzidine. The analyte, because it mimics the enzyme peroxidase, causes a reaction between the indicator and organic hydroperoxide which yields a color, the intensity of which is a barometer of the analyte concentration. In light of the unmistakable teachings of peroxidase activity shown by Co(III) complexes, one skilled in the art would clearly not expect such a substance to be compatible with the peroxide/indicator system. Clearly, if one incorporates an analyte into the very reagent formulation designed to change color in the presence of that analyte, it is to be expected that false positive results would be obtained. These conclusions notwithstanding, it has been surprisingly found that the peroxidatively active Co(III) complexes not only fail to give false positive results, but they actually improve the reagent system, making it even more dependable, i.e., less sensitive to the inaccuracies caused by ascorbate interference.